The completion of the Human Genome Project presents us with the tremendous possibility of understanding the function of genes and the role of proteins in cellular pathways. Methods for visualizing and quantifying protein-protein complexes in real time in living cells would enable biology-based drug discovery on a large scale. These methods could potentially be used to elucidate altered cellular pathways that underlie major chronic diseases including cancer, cardiovascular, autoimmune and neurodegenerative conditions; to map targets into pathways; to perform high-throughput screening of small-molecule libraries; and to identify on- pathway and off-pathway effects of known and unknown drugs. Existing assays (e.g. Protein fragment Complementation Assay, PCA) used for visualization and quantification of protein-protein interactions in live cells represent a step toward pathway-based drug discovery. A major drawback of such assays is the use of large reporter fluorescent proteins that may interfere with and/or sterically hinder the target protein's function or generate false positive or negative results. Inherent complexities with use of large reporter proteins make the design, development and validation of such assays time-consuming and expensive, limiting the application and impact of this technology. To overcome the technical difficulties associated with existing approaches, we will develop a novel method for in vivo measurement of protein-protein complexes that detects signals from a small fluorophore that has been site-specifically and biosynthetically incorporated into the protein of interest. We will generate cell lines that express an orthogonal aminoacyl transferase/amber suppressor tRNA pair that specifically incorporates a BODIPY-amino acid derivative into the elongating target protein sequence at positions in the target mRNA designated by the amber stop codon. Fluorescence polarization measurements will be used to detect the interaction of the fluorophore-labeled target protein with a second protein. In the Phase I studies, as a model assay we will establish a cell-based assay for measuring the FK506-mediated dimerization of FKBP12 with calcineurin (CN). In Aim 1 we will establish the expression of FKBP12 and CN in mammalian cells. In Aim 2, we will optimize the expression and characterize the fluorescence polarization of a series of BODIPY-FKBP12. In Aim 3, we will optimize co-expression of BODIPY-FKBP12 and CN and determine which BODIPY-FKBP12 proteins are optimal for measuring FK506-mediated dimerization using fluorescence polarization. We believe our approach will allow the end-user to rapidly develop cell-based assays for quantifying protein-protein interactions that can be used to map intracellular pathways and screening of small-molecule protein-protein interaction modulators.